The long term goal of this project is to improve our understanding of molecular mechanisms of drug resistance and drug action in Mycobacterium tuberculosis. Such knowledge will be useful for rapid detection of drug resistant M. tuberculosis and rational design of new antituberculous drugs. Drug resistant tuberculosis is on the increase recently and poses serious threat to the treatment of the disease. This project proposes to study the molecular basis of pyrazinamide resistance in M. tuberculosis. the specific aims of the project are: 1) To determine how acid pH potentiates pyrazinamide activity; 2) To characterize mutations in the pyrazinamidase gene and assess the role of these mutations in pyrazinamide resistance by transformation studies; 3) To develop a PCR-based test for rapid detection of pyrazinamide resistance in clinical isolates of M. tuberculosis. The health relatedness of this project lies in the possible development of a rapid test for direct detection of pyrazinamide resistance in clinical isolates of M. tuberculosis and also in the design of new antituberculous drugs based on the understanding of the drug action. Two hypotheses about the role of acid pH in potentiating pyrazinamide activity will be tested in Specific Aim 1. The first hypothesis that acid pH may enhance the uptake or transport of pyrazinamide in M. tuberculosis will be tested by monitoring the derivative of radioactive 14C-pyrazinamide by thin layer chromatography (TLC). the second hypothesis that acid pH may activate the pyrazinamidase gene or the enzyme itself will be tested by using reporter gene constructs and in vitro enzyme assays. Potential mutations in the pyrazinamidase gene among pyrazinamide-resistant M. tuberculosis strains will be analyzed by PCR sequencing. The role of the mutations in the pyrazinamidase gene will be assessed by transformation of resistant strains with a functional pyrazinamidase gene. Furthermore, a rapid test will be developed for detection of pyrazinamide resistance in M. tuberculosis by PCR-SSCP. Such a rapid DNA-based test is particularly important in view of the recent increase of drug resistant tuberculosis and the difficulty of current pyrazinamide susceptibility testing.